Skeletal muscle tissue dissociation by enzymatic digestion and isolation of FAPs by FACS with anti-CD140a (PDGFRA)–APC (eBioscience, 17-1401-81) and anti-Ly6A/E (SCA-1)-v450 (BD Biosciences, 560653) was conducted as previously described (6 (link), 12 (link), 18 (link), 35 (link)), with the following modification: magnetic depletion of CD31+ and CD45+ cells prior to FACS was omitted, and CD31+ and CD45+ cells were detected with anti-CD31-bv711 (BD Biosciences, 740690; 1:800) and anti-CD45-bv711 (BD Biosciences, 563709; 1:500) antibodies. Sorting was performed on a FACSAria II (BD Biosciences) equipped with 405, 488, 561, and 633 nm lasers.
FACS-isolated FAPs were grown on tissue culture flasks (Nunc) in DMEM (Thermo Fisher Scientific, 11995065) with 50 U/mL penicillin and 50 μg/mL streptomycin (Pen/Strep; Gibco) and 20% premium FBS (Atlanta Biologicals, lot C19032) as previously described (6 (link), 12 (link)). FAPs were maintained at 37°C in a humidified atmosphere at 5% CO2. Media were changed every other day. All experiments utilized FAPs that had undergone less than 7 population doublings (approximately 3 passages).
FACS-isolated FAPs were grown on tissue culture flasks (Nunc) in DMEM (Thermo Fisher Scientific, 11995065) with 50 U/mL penicillin and 50 μg/mL streptomycin (Pen/Strep; Gibco) and 20% premium FBS (Atlanta Biologicals, lot C19032) as previously described (6 (link), 12 (link)). FAPs were maintained at 37°C in a humidified atmosphere at 5% CO2. Media were changed every other day. All experiments utilized FAPs that had undergone less than 7 population doublings (approximately 3 passages).
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